Growth plate development is a critical early step in endochondral bone formation. Abnormalities in the growth plate are linked to skeletal diseases and cause growth retardation. Levels of systemic Ca2+ play an important role in the production of matrix proteins and the calcification of cartilage matrix. In vitro studies show that changes in the extracellular Ca2+ concentration ([Ca2+]o) modulate the expression of markers of differentiation in cultured chondrocytes. We have found that growth plate chondrocytes can sense (Ca2+)o and couple them to changes in proteoglycan accumulation, gene expression and mineralization. We hypothesize that growth plate chondrocytes express extracellular Ca2+-sensing mechanisms that regulate cell differentiation and cartilage mineralization and that Ca2+ receptors (CaRs) mediate these Ca2+-sensing functions. We propose three Aims to address this hypothesis: (1) we will assess whether changes in the (Ca2+)o regulate growth plate chondrocyte differentiation using stage-specific protein markers and cartilage mineral production; (2) we will determine whether the extracellular Ca2+-sensing mechanism in growth plate chondrocytes activates signal transduction pathways typically coupled to CaRs at concentrations of ionic and organic CaR agonists that are characteristic of CaRs; (3) we will ascertain whether changes in proteoglycan production, mineralization, and intracellular Ca2+ responses induced by high (Ca2+)o are mediated by CaRs by overexpressing antisense or signaling-efficient CaR mutants in cultured growth plate chondrocytes and testing the effects of CaR agonists in growth plate chondrocytes from CaR knock-out mice. These studies will advance our understanding of the molecules and pathways responsible for Ca2+-sensing in chondrocytes and offer the potential for therapies directed at treating chondrodysplasias and arthropathies due to disturbances in systemic and local Ca2+ availability.